2.4. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The microdilution assay was performed using 96-well plates as described by Eloff, 1998 with slight modifications (Eloff, 1998). Briefly, 100 µL of fresh MHB was added into wells in row B to H in column 1 to 11 and 200 µL of fresh media at column 12 in row A to D as a blank control. 100 µL plant extract working solutions were added in row A and B in column 1 to 9 in triplicate and 100 µL antibiotic at column 10 to 11. A two-fold serial dilution was performed by transferring 100 µL from row B to C in column 1 to 11 using a multichannel pipette. The same step was performed until row H and the excess 100 µL mixture from row H was discarded. 100 µL of diluted standardized bacterial suspension was added into the wells of row A to H in column 1 to 11 as well as in column 12 from row E to H as a growth control. The final volume in each well was 200 µL. The concentration of the extracts was ranged between 800 and 6.25 µg/mL. The final volume in the wells was 200 µL and the DMSO concentration was less than 1% (v/v).

The plate was incubated for 18 to 22 hrs at 37 ˚C. After the incubation period, 50 µL of freshly prepared INT solution wasadded into all wells except column 3, 6, and 9. The MIC plate was then re-incubated for two hrs at 37 °C. The MIC is defined as the lowest drug or extract concentration that prevented a colour change of the INT from colourless to pinkish. The pinkish colour indicated bacterial growth. On the other hand, 10 µL of the mixture from column 3, 6, and 9 were inoculated into MHA plates to determine the MBC. The plates were incubated at 37 °C for 18 to 22 hrs in an incubator (Memmert, Germany). MBC was determined as the lowest concentration of drug or extract which did not produce any bacterial colonies on the agar plate. The MIC and MBC were determined from three independent experiments performed in triplicates.